Volume 309, number 3, 421--423

FEBS 11551 © 1992 Federation of Euro~an Biochemical Societies 00145793/92/$5.00

Site-directed mutagenesis of active-site-related residues in acetylcholinesterase

September 1992


Prcsence of a glutamic acid in the catalytic triad Nathalie D u v a P , S u z a n n e B o n ~, Israel g i l m a n b, Joel S u s s m a n b a n d Jean Massouli6" aLaboratoire de Neurobiologieo CNRS UA 295, Ecole Normale gupdrieure, 46 rue d'Uhn, 75005 Paris, France and bThe Weizmann Institute of Science, Rehot,ot 76100, Israel Received 30 June 1992 Site-directed nautagen¢fi5was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu.327, together with His-440 and Set-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfeetioa of appropriately mutated DNA into COS cells showed that the mutation of Asp-326~Asn had little effect on catalytic activity or the molecular forms expressed, sulggestin8no crucial structural or functional role for this residue. Mutation of Glu-327 to Gin or to Asp led to an inactive product. These results support the conclusionsof the structural analysis for the two acidic residues. Acetylcholinesterase; Catalytic triad; Site-directed mutagenesis; C O S cell

1. I N T R O D U C T I O N The principal biological role of acetylcholinesterase (ACHE, EC is the termination of impulse transmission at cholinergic synapses by hydrolysis of the neurotransmitter, acetylcholine [1]. In keeping with this functional requirement, AChE is an extremely rapid enzyme, operating, under optimal conditions, at a rate approaching that at which substrate diffusion becomes rate-limiting [2]. Kinetic studies suggest that the catalytic mechanism of AChE is rather complex. The chemical steps of acylation and deacylation of the active-site serine are preceded by a conformational step [3]. In addition, the influence of the isotopic ratio in D20/H.~O solvents indicates the transfer of a single proton, suggesting that a charge relay system, such as that believed to operate in the serine proteases [4], which involves several protons, is not functional in this case [2,5]. The recent determination of the three-dimensional atomic structure of AChE from Torpedo californicaper. mits, for the first time, direct visualization of its active site [6]. In particular, this structure shows that the active-site serine (Set-200) forms a catalytic triad similar to that observed in chymotrypsin [4]. The participation of His-440 in such a triad had previously been inferred, Correspondence address: J. Ma~5ouli~,Laboratoir¢ de Neurolaiolo~ie, CNRS UA 295, EcoleNormale SupC~ricure,46 rue d'Ulm, 75005 Paris, France.

Published by Elsevier Science .Pi~blisiters B.V.

since it is the only histidine residue conserved within the family of cholinesterases and homologous enzymes [7], and its mutagenesis to glutamine totally abolished catalytic activity [8]. The three-dimensional structure indeed placed His-440 within hydrogen-bonding distance of Ser2000~,nm~ [6]. The acidic member of the thud, Glu327, had not been identified previously. In all serine hydrolase structures solved hitherto, this residue was an aspartic acid. Torpedo AChE [6], and the structurally related lipas¢ from Geotrichumcandidum, the structure of which was also solved only recently [9], are the first cases reported in which Asp is replaced by Glu. It is interesting that Glu-327 is replaced by an aspartic residue in some of those esterases which present a clear homology of their primary structure with that of the cholinesterases [7]. In addition, this residue is immediately preceded by an aspartic acid in all known primary structures of cholinesterases. In the present study we report the effect of site-directed mutagenesis o f Asp- 326 and Glu-327 on the catalytic activity of TorpedoACHE. 2. M A T E R I A L S A N D M E T H O D S in order to mutageniz¢ aminoacids 326 and 327 of Torpedo ACHE, we amplified a restriction i'~gment (mw.leotld¢~ 1017-1695 of the coding sequence, numbered as in [10]) in the expression vector CDM8AChET [7,11], using a mutagenizing oligonueleotide primer and a common primer. The mumgenizingprimers were based on the followin~ ~eq~ienee: 5"-GAAGACTAGATC'I'TACTGGGAGTCAACAAGG*ACG*AG*GGCTCGT-3'.


Volume 309, number 3


This sequence corresponds to nucleotides 1017-1060, in the coding orientation; it contains a Balll r~triction site (underlined). Either one or two of the O nucleotides marked by asterisks (I 048, 105 i, and 1053) were replaced by other nucleotldgs: G ~as~-Cfor Glu-327---~Asp, G ~0~.C for Glu-327~GIn, G ~ - A for Asp-326~Asn, and a combination of the last two changes for the double mutation Asp.326~Asn/Olu. 327~Gln. The common primer was in tile antisense orientation, and was complementary to nucleotides 1671-1695 of 2AChE 15-2: 5'TCG'I'FTGGG1-rTCCAOTCTTTGCO-3'. The amplification (PCR) reaction mixture contained 500 n8 of the common and mutagenizing primer, 5 n8 CDM8-AChEv DNA, 200

September 1992 C O S cells + CDM8-AChET i


~ o . , . _ _ _



. . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . .

m M dNTP, one unit of Taq polymcra~e (Bioprobe Systems, Montrcuil-sous-Bois,France), 5/xl of 10)

Site-directed mutagenesis of active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad.

Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The...
257KB Sizes 0 Downloads 0 Views